POS0406 CHARACTERISATION OF ENHANCER RNAs IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS
Authors: L. Moser, M. Houtman, O. Distler, B. Maurer, C. Ospelt, K. Klein
Journal: Annals of the Rheumatic Diseases (2022)

Background Rheumatoid arthritis synovial fibroblasts (SF) exhibit a prolonged histone 3 lysine 27 acetylation (H3K27ac) after stimulation with TNF, leading to a sustained inflammatory response. Enhancer RNAs (eRNAs) are short-lived non-coding RNAs transcribed from cell type-specific H3K27ac-marked enhancers that facilitate the transcription of their linked coding genes. The interrelation of eRNAs and their linked coding genes in SF has not been studied yet. Objectives To analyze the expression and regulation of inflammatory eRNAs in SF. Methods The expression of eRNAs in SF (n=9), stimulated with TNF (24h; 10 ng/µl) or left untreated, was detected by cap analysis of gene expression followed by sequencing (CAGEseq). To further investigate eRNA expression in SF, the expression of selected eRNAs and their linked coding genes ( CXCL1 , CCL20 , IL6 , CCL2 , CXCL12 , IL8 ) was analyzed by real-time PCR in SF that were stimulated with TNF (1, 3, 6, 24h; 10 ng/µl), IL1 (1, 24h; 1 ng/ml), or the Toll-like receptor agonists Pam3 (1, 24h; 1 ng/ml), pIC (1, 24h; 10 μg/ml) and LPS (1, 24h; 100 ng/µl). Samples containing the untranscribed RNA were measured in parallel. To study eRNA regulation, SF were treated with the bromodomain inhibitor I-BET (1 µM; 24h), or silenced for the histone acetyltransferases CBP and p300 by transfection of antisense LNA gapmeRs (12.5 nM) prior to stimulation with TNF (1, 24h). Results We have selected four potential eRNAs for CCL2 and IL8, three for CXCL12, two for IL6 and CXCL1 and one for CCL20 from CAGEseq analysis. They were located upstream (eCCL2#1, eCCL2#2, eCXCL1#2, eCXL12#3, eIL6#1 + 2, eIL8#1-4), downstream (eCCL2#3, eCCL2#4, eCXCL1#1, eCXL12#1) and intronic (eCCL20, eCXL12#2) at distances between 300 bp to 35.6 kb relative to the transcription start sites of the corresponding coding genes. None of the eRNAs was present in all nine samples in CAGEseq data sets, indicating a patient-dependent variability in eRNA expression. The majority of eRNAs were not detected in unstimulated SF, with the exception of eRNAs for CXCL12. By performing TNF time course experiments (Figure 1), we have detected different patterns of eRNAs: (a) eRNAs, that peaked at 1h (eCCL20, eIL8#2, eCCL2#1), (b) at 6h (eCXCL1#1), (c) or at 24h (eIL8#1, eIL8#3, eIL8#4, eCXCL1#2) after stimulation, (d) eRNAs that were stably expressed over the time points (eCCL2#2, 3, 4), and (e) eRNAs that were down regulated by TNF stimulation (eCXCL12#1, eCXCL12#3). All inflammatory stimuli induced eRNA expression in SF, with LPS and IL1, followed by TNF, being the most potent inducers of eRNAs. I-BET suppressed the TNF-induced expression of all eRNAs tested. The effects on the expression of eRNAs after silencing of p300 but not of CBP mirrored to a large extent those of the respective coding genes. Figure 1. Conclusion In SF, the expression of some eRNAs is maintained for up to 24h, contradicting previous reports that eRNAs are short-lived. Our data suggest that different eRNAs orchestrate the early and sustained expression of cytokines and chemokines in SF. eRNA expression is controlled by p300 and BET bromodomain proteins. Disclosure of Interests Larissa Moser: None declared, Miranda Houtman: None declared, Oliver Distler Consultant of: Abbvie, Britta Maurer Speakers bureau: Boehringer-Ingelheim, Consultant of: Novartis, Boehringer Ingelheim, Janssen-Cilag, Grant/research support from: AbbVie, Protagen, Novartis Biomedical Research, Caroline Ospelt: None declared, Kerstin Klein Grant/research support from: Novartis Foundation for biomedical research (2019)